Project 1- Abstract Project 1 focuses on the regulation of the ?3 integrins on cells exposed to blood. In the past funding period, evidence has been developed to demonstrate a previously underappreciated role of the kindlins in integrin function by showing that they are essential regulators of integrin activation. Kindlin-2 (K2) and kindlin-3 are known to play a particularly prominent role in controlling the functions of the two ?3 integrins, ?IIb?3 and ?V?3, on platelets, endothelial cells and leukocytes. This project focuses on K2, and the preliminary data support the hypothesis that K2 regulates: 1) integrin inside-out responses as exemplified by its control of ?V?3 activation; 2) outside-in signaling via its regulation of cell spreading; and 3) integrin independent responses as exemplified by its regulation of membrane proteins expression. Consequently, K2 plays important and diverse roles in vascular biology, including the capacity to regulate: a) ?V?3 activation, which controls angiogenesis; b) outside-in signaling via a previously unrecognized and direct interaction with actin; c) integrin independent responses as demonstrated by its selective regulation of trafficking of a subset of membrane proteins in endothelial cells, which controls hemostasis, and d) interaction with proteins of adherens junctions, which controls vascular permeability. To test the elements of this hypothesis will require an in-depth understanding of K2 structure-function relationships, and animal models in which the functions of K2 can be interrogated in various biological settings. To probe the structure-function relationships of K2, mutational analyses driven by NMR studies will be performed on specific sites in the molecule that regulate its biological activities. For in vivo analyses, unique mouse strains developed in the laboratory, floxed K2 and mutant K2QW/AA knock-in mice which are unable to bind integrins, will be used. The specific aims of the project are: 1) To delineate the function of K2 in vivo in which K2 has been selectively deleted from endothelial cells, express the K2WQ/AA mutant or K2+/- mice, which has been used successively and extensively by us to identify previously unknown functions of K2. Angiogenesis, hemostasis, trafficking of membrane proteins and changes in vascular permeability will be examined in these mouse strains. 2) To resolve the molecular mechanisms, which allow K2 to orchestrate its integrin-dependent and independent functions. The basis for the preference of kindlins for the ?3 integrins, the specificity of clathrin binding motif in K2, and the previously unappreciated role of the small C-terminal segment of K-2 in integrin activation will be determined. A particularly novel aspect of these studies will be to pursue the lead that K2 binds to a membrane proximal site to alter the transmembrane region of ?3 and influence the activation process. 3) To determine the location, specificity and functional consequences of the direct interaction of K2 with actin. These studies will establish how K2 functions at a molecular and cellular level and how these roles manifest in the intact organism.